1614 Chem. Res. Toxicol., Vol. 19, No. 12, 2006
Beyerbach et al.
1
0.37 mmol) were kept in anhydrous pyridine (3 mL). After 6 h at
100 °C, they were evaporated to dryness and coevaporated with
toluene, and 235 mg of a white solid was obtained. Purifica-
tion was by flash chromatography on silica gel with CHCl3/MeOH
) 9:1, yielding 4M-AcdG (90 mg, 0.204 mmol, 55%) as a white
solid.
4M-dA. H-NMR (250 MHz, [d6]DMSO): δ [ppm] ) 2.28 (s,
3 H, CH3), 2.36-2.43 (m, 1 H, 2′-H), 2.75-2.81 (m, 1 H, 2′-H),
3.57-3.66 (m, 2 H, 2 × 5′-H), 3.93 (m, 1 H, 4′-H), 4.47 (m, 1 H,
3′-H), 5.04 (m, J ) 5.5 Hz, 1 H, OH), 5.38 (d, J ) 4.1, 1 H, OH),
6.47 (‘t’, J ) 6.7 Hz, 1 H, 1′-H), 7.16 (d, J ) 8.3 Hz, 2 H, arom
2-H, 6-H), 7.52 (d, J ) 8.3 Hz, 2 H, arom H-3, H-5), 8.69 (s, 1 H,
2-H or 8-H), 8.70 (s, 1 H, 8-H or 2-H), 10.06 (br. s, 1 H, NH),
11.73 (br. s, 1 H, NH).
This was suspended in CHCl3/MeOH ) 1:1 (10 mL), 5 M
sodium hydroxide (250 µL, 10 drops from a Pasteur pipet) was
added, and after 5 min, the solution was neutralized with Dowex
50-X8 (20-50 U.S. mesh) [H+ - form]. The Dowex was filtered
off, washed with 10% formic acid (30 mL), and the solution was
evaporated in Vacuo, yielding a white solid, which was pure
4M-dG (72 mg, 0.18 mmol, 49%) in the form of white crystals.
4M-AcdG. TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.33.
4M-dG. TLC (silica gel, CHCl3/MeOH ) 8:2): Rf ) 0.
4M-AcdG. FAB-MS (NBA): m/z (%) ) 529 (43, [MH +
2Na]+), 507 (84, [MH + Na]+), 485 (42, [M + H]+), 307 (98,
[MH + Na - (diacetyl + dR)]+), 285 (100, [MH - (diacetyl +
dR)]+).
4M-dA. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 20.8
(CH3), 40.2 (CH2-2′), 61.9 (CH2-5′), 71.0 (CH-3′), 84.2 (CH-1′),
88.4 (CH-4′), 119.8 (arom. CH-2, CH-6), 120.8 (C-5), 129.7 (arom.
CH-3, CH-5), 132.5 (arom. C-4), 136.2 (arom. C-1), 142.7
(CH-8), 151.1 (CH-2), 150.3 150.6 151.3 (C-4, C-6, CO).
4M-dA. UV (HPLC-run: MeOH/ammonium formate pH 5.5):
λ nm (E): 236max (0.198), 250min (0.174, 53%), 281max (0.330,
100%).
Synthesis of N4-(N-p-Methylphenyl-carbamoyl)-2′-deoxycy-
tidine (4M-dC). 4MPI (98 mg, 0.74 mmol) + AcdC (115 mg,
0.37 mmol) were in anhydrous pyridine (3 mL). After 3 h at 100
°C, they were evaporated to dryness, coevaporated with toluene,
and 225 mg of a white solid was obtained. (Using flash chroma-
tography on silica gel with CHCl3/MeOH ) 97:3 no good separation
was achieved.) The solid was taken up in CHCl3/MeOH ) 1:1 (10
mL), 5 M sodium hydroxide (250 µL, 10 drops from a Pasteur
pipet) was added, and after 5 min, the solution was neutralized
with Dowex 50-X8 (20-50 U.S. mesh) [H+ - form]. The Dowex
was filtered off, and the solution was evaporated in Vacuo, yielding
a white solid (110 mg), which was purified by flash chromatography
on silica gel with CHCl3/MeOH ) 85:15, yielding 4M-dC (88 mg,
66%) in the form of white crystals.
4M-dG. FAB-MS (glycerol): m/z (%) ) 401 (39, [M + H]+),
285 (100, [M - dR]+), 115 (43, [dR]+).
4M-dG. ESI-MS: m/z) 423 [M + Na]+, 401 [M + H]+, 316
[(dG + CO) + Na]+, 307 [(M - dR) + Na]+, 294 [(dG + CO) +
H]+, 285 [(M - dR)] + H]+, 200 [(guanine + CO) + Na]+, 178
[(guanine + CO) + H]+, 174 [guanine + Na]+, 152 [guanine +
H]+, 139 [dR + Na]+, 117 [dR + H]+.
4M-dG. 1H-NMR (250 MHz, [d6]DMSO): δ [ppm] ) 2.28 (s,
3 H, CH3), 2.28-2.33 (m, 1 H, 2′-H), 2.56-2.66 (m, 1 H, 2′-H),
3.52-3.63 (m, 2 H, 2 × 5′-H), 3.87 (m, 1 H, 4′-H), 4.39 (m, 1 H,
3′-H), 4.97 (br. s, 1 H, OH), 5.33 (br. s, 1 H, OH), 6.22 (‘t’, J )
6.6 Hz, 1 H, 1′-H), 7.16 (d, J ) 8.2 Hz, 2 H, arom 2-H, 6-H), 7.39
(d, J ) 8.31 Hz, 2 H, arom. 3-H, 5-H), 8.17 (s, 1 H, 8-H), 9.69
(br. s, 1 H, NH), 10.25 (br. s, 1 H, NH), two signals for NH are
missing. This was also reported for a similar adduct by Tamura
et al. (22) .
TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.70 (4M-AcdC),
0.15 (4M-dC).
4M-dC. FAB-MS (NBA): m/z (%) ) 361 (28, [M + H]+), 245
(100, [MH - dR]+).
4M-dC. ESI-MS: m/z ) 383 [M + Na]+, 361 [M + H]+, 267
[(M - dR) + Na]+, 250 [dC + Na]+, 245 [(M - dR) + H]+,
160 [(cytosine + CO) + Na]+, 134 [cytosine + Na]+, 112 [cytosine
+ H]+.
4M-dG. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 20.1
(CH3), 40.2 (CH2-2′), 61.9 (CH2-5′), 70.9 (CH-3′), 83.5 (CH-1′),
88.2 (CH-4′), 119.7 (arom. CH-2, CH-6), (117-120: C-5 signal
not seen), 129.8 (arom. CH-3, CH-5), 132.9 (arom. C-4), 135.5
(arom. C-1), 137.4 (CH-8), 148.9, 152.9 (C-4, C-2, C-6, CO,
between ca. 148-160, two signals are missing because of low signal
intensities).
4M-dG. UV (HPLC-run: MeOH/ammonium formate pH 5.5):
λ nm (E): 259 max (0.534, 100%), 277 shoulder (0.424, 79%)
Synthesis of N6-(N-p-Methylphenyl-carbamoyl)-2′-deoxyad-
enosine (4M-dA). 4MPI (99 mg, 0.746 mmol) + AcdA (125 mg,
0.37 mmol) were in anhydrous pyridine (3 mL). After 2.5 h at 100
°C, they were evaporated to dryness, coevaporated with toluene,
and 218 mg of a white solid was obtained. This was dissolved in
CHCl3, and MeOH was added to precipitate the 4M-AcdA. The
mixture was centrifuged, and the supernatant was decanted. Again,
the white solid was taken up in CHCl3 and precipitated with MeOH.
The precipitate was dried thoroughly, yielding 4M-AcdA.
The solid was taken up in CHCl3/MeOH ) 1:1 (10 mL), 5 M
sodium hydroxide (250 µL, 10 drops from a Pasteur pipet) was
added, and after 10 min, the solution was neutralized with Dowex
50-X8 (20-50 U.S. mesh) [H+ - form]. The Dowex was filtered
off, and the solution was evaporated in Vacuo yielding a white solid,
which was pure 4M-dA (84 mg, 0.218 mmol, 59%) in the form of
white crystals.
1
4M-dC. H-NMR (250 MHz, [d6]DMSO): δ [ppm] )2.01-
2.12 (m, 1 H, 2′-H), 2.26 (s, 3 H, CH3), 2.26-2.36 (m, 1 H, 2′-H),
3.55-3.68 (m, 2 H, 2 × 5′-H), 3.88 (m, 1 H, 4′-H), 4.26 (m, 1 H,
3′-H), 5.09 (br. s, 1 H, OH), 5.30 (br. s, 1 H, OH), 6.16 (‘t’, J )
6.3 Hz, 1 H, 1′-H), 6.50 (d, J ) 6.9 Hz, 1 H, 5-H), 7.14 (d, J ) 8.2
Hz, 2 H, arom. 2-H, 6-H), 7.39 (d, J ) 8.2 Hz, 2 H, arom. 3-H,
5-H), 8.28 (d, J ) 7.5 Hz, 1 H, 6-H), 10.22 (br. s, 1 H, NH), 11.28
(br. s, 1 H, NH).
4M-dC. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 22.8
(CH3), 41.1 (CH2-2′), 61.3 (CH2-5′), 70.3 (CH-3′), 86.5 (CH-1′),
88.2 (CH-4′), 95.3 (CH-5), 119.6 (arom. CH-2, CH-6), 129.7 (arom.
CH-3, CH-5), 132.7 (arom. C-4), 136.0 (arom. C-1), 144.3
(CH-6), 151.6 (CO), 153.9 (C-2), 162.6 (C-4).
4M-dC. UV (HPLC-run: MeOH/ammonium formate pH 5.5):
λ nm (E): 232max (0.528, 100%), 262min (0.261, 49%), 295max
(0.403, 76%).
Reaction of Isocyanates with DNA. The isocyanate was added
to DNA (5 mg) in water (2 mL) and THF (2 mL, HPLC quality).
After an incubation time of 2 h at 37 °C, the reaction mixture was
extracted with diethyl ether and ethyl acetate (3-4 × 6-8 mL).
After centrifugation (3000g), the aqueous solution was transferred
with a Gilson pipet into another tube. The solution was buffered
with 50 mM Bis-tris and 1 mM MgCl2 at pH 6.5. Per 1 mg of
DNA and 1 mL of solution, the following three enzymes were
added: 24 units of nuclease P1 (310 units/mg solid in 260 µL of
1 mM ZnCl2 ) 1.2 units/µL), 2.4 units of alkaline phosphatase
(50 units in 100 µL of water ) 0.5 units/µL), 0.3 units of acid
phosphatase (10 mg of solid (8 units) in 80 µL of water ) 0.1
units/µL). The mixture was incubated for 8 h at 50 °C. After
denaturation of the enzymes at 100 °C for 5 min, the enzymes were
removed by centrifugation.
4M-AcdA. TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.72.
4M-dA. TLC (silica gel, CHCl3/MeOH ) 8:2): Rf ) 0.37.
4M-AcdA. FAB-MS (NBA): m/z (%) ) 469 (64, [M + H]+),
269 (100, [MH - (diacetyl + dR)]+).
4M-dA. FAB-MS (NBA): m/z (%) ) 385 (55, [M + H]+), 269
(100, [MH - dR]+).
4M-dA. ESI-MS: m/z ) 407 [M + Na]+, 385 [M + H]+, 300
[dA + CO + Na]+, 291 [(M - dR) + Na]+, 269 [(M - dR) +
H]+, 184 [(adenine + CO) + Na]+, 162 [(adenine + CO) + H]+,
158 [adenine + Na]+, 139 [dR + Na]+, 136 [adenine + H]+, 117
[dR + H]+.
4CPI Reaction with DNA. CHCl3 (4 mL) was added, and the
mixture was extracted. Centrifugation at 3000g for 5 min yielded